Artificial neuronal microtissues provide exogenous axons for non-instantaneous nerve fusion and rapid neuromuscular recovery

Abstract

In various aspects and embodiments, the invention provides a tissue engineered neuromuscular interface comprising: an extracellular matrix core comprising a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons extending at least a portion along the extracellular matrix core, the population of neurons being selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 209,639, filed June 11, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Award W81XWH-16-1-0796 from the Army and Merit Award I01-BX003748 provided by the U.S. Government as represented by the Department of Veterans Affairs. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Peripheral nerve injury (PNI) is estimated to be present in 3% of trauma cases and up to 5% when plexus and root avulsion injuries are included. Over 550,000 PNI procedures are performed annually in the United States. Despite recent advances in neurosurgery, it is estimated that only 50% of patients achieve satisfactory functional recovery. Although several factors influence successful regeneration, delayed surgical repair is considered the most important factor for poor functional recovery. After injury, axons of distal nerves undergo Wallerian degeneration. Dedifferentiated Schwann cells temporarily form cylindrical pro-regenerative structures called Büngner's zones that promote axonal regeneration and reinnervation of target muscles. Over time, prolonged denervation due to loss of axonal contact secondary to delayed surgical repair leads to deterioration of the Büngner's zones, attenuating the potential for muscle reinnervation and ultimately functional recovery. Unfortunately, delayed surgical repair is common as surgeons utilize a "wait and see" approach to determine the potential for spontaneous recovery. Thus, there is an unmet clinical need for new strategies that can extend the optimal surgical window and improve the chances of meaningful recovery. Accordingly, there is a need in the art for improved methods of nerve repair. The present disclosure addresses that need. Summary of the Invention

[0004] [Brief description of the drawings]

[0005] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there is shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0006] [Figure 1]Figures 1A-1F show the fabrication and characterization of tissue engineered neuromuscular interfaces (TE-NMIs). Figure 1A: TE-NMIs are anatomically inspired bioengineered pathways with distinct neuronal populations spanning sensory, motor, or both motor and sensory axonal tracts within a protective biomaterial enclosure. The modular TE-NMI fabrication process allows for the construction of microcolumn hydrogels with various diameters, neuronal cell sources, or biomaterial outer enclosures. Figure 1B: Representative phase images and (B') confocal images of a sensory TE-NMI with an outer diameter of 2 mm and an inner diameter of 1 mm, labeled with the neuronal / axonal marker Tuj1 (green) and counterstained with Hoechst (blue) to identify nuclei. Figure 1C: Representative confocal images of a motor TE-NMI with an outer diameter of 350 μm and an inner diameter of 180 μm at 7 days in vitro (DIV) that was virally transduced to express green fluorescent protein (GFP). Figure 1D: At 14 in vitro days, phase imaging revealed two distinct populations of motor neurons (MNs) spanning their axons. (D') High-resolution confocal imaging revealed distinct regions of motor neurons / axons labeled for Tuj1, the motor neuron specific marker ChAT, and Hoechst. Figure 1E: Representative confocal images of constructs with agarose or agarose-gelatin composite (AGX) outer enclosures at 7 DIV. Figure 1F: The length of neurites within each biomaterial enclosure was compared at 1 and 3 DIV. Mean values ​​were compared using two-way ANOVA. Error bars represent standard error. *p<0.05; **p<0.01; ****p<0.0001. Scale bars: Figure 1C-1D 500 μm, zoomed in: 100 μm. Figure 1E 500 μm. [Diagram 2]Figures 2A-2G show TE-NMI survival, growth, and integration with otherwise denervated nerves. Figure 2A: Schematic illustrating the chronic host axotomy surgery model and experimental groups, including transplantation of an acellular column, one TE-NMI, or two TE-NMI. Acellular controls were also transplanted as negative controls. We hypothesized that TE-NMI would extend axons that interact with Schwann cells in the otherwise denervated distal nerve. Figure 2B: Intraoperative photographs showing that TE-NMI can be microinjected into the nerve. Figure 2C: Representative images of microinjected TE-NMI at 2 weeks post-implantation, visualized following optical clearing and multiphoton microscopy. Robust TE-NMI neurons and axons (GFP) were seen within the lumen protected from host cells invading the graft area. Figure 2D: To assess whether TE-NMI axons extended and interacted with Schwann cells within the otherwise denervated nerve, nerve cross sections taken 5 mm distal to the graft site were labeled for Schwann cells (S100) and TE-NMI axons (GFP). Figure 2E: High-resolution image showing an example of a GFP+ TE-NMI axon extending through aligned Schwann cells resembling a Bungener zone. Figure 2F: Greater growth was seen with GFP distal to the 2 TE-NMI than 1 TE-NMI. Figure 2G: Distal to the graft site, there was increased coverage with S100 in the 2 TE-NMI group. These findings suggest that TE-NMI may interact with and potentially influence host S100+ Schwann cells within the denervated distal nerve at 6 weeks post-graft in the host nerve axotomy model. Error bars represent standard error. Means were compared using one-way ANOVA followed by Tukey's post-hoc test. *p<0.05. Scale bars: Fig. 2C: 100 μm, Fig. 2D: 100 μm, Fig. 2E: 5 μm. [Diagram 3]Figures 3A-3D show evoked muscle responses 16 weeks after TE-NMI implantation in a chronic host axotomy model. Figure 3A: Schematic illustrating the surgical model, implantation paradigm, and outcome measures. In a chronic host axotomy model, a mixed motor-sensory TE-NMI was fixed to the common peroneal nerve. Evoked muscle responses were recorded 16 weeks after implantation following percutaneous stimulation of the common peroneal nerve innervating the distal target tibialis anterior muscle. Figure 3B: Representative confocal images of mixed motor-sensory TE-NMI containing neuronal populations transduced to express TD-tomato (motor, red) or GFP (sensory, green). Figure 3C: Reproducible and robust waveforms were evoked in the TE-NMI group compared to irregular / absent recordable waveforms in the no implant or microcolumn only control groups. Figure 3D: Larger mean amplitude evoked muscle responses were observed in the TE-NMI group compared to controls. These findings suggest that TE-NMI functionally integrates with denervated muscles and preserves electrophysiological muscle responses 16 weeks after chronic axotomy. Error bars represent standard error. Means were compared using one-way ANOVA followed by Tukey's post-hoc test. **p<0.01. [Figure 4]Figures 4A-4I show non-immediate axonal fusion via freshly transected TE-NMI axons at an otherwise denervated distal nerve. Figure 4A: Schematic illustrating the surgical model, non-immediate nerve fusion paradigm, and outcome measures. At 20 weeks after graft and host chronic nerve axotomy, the TE-NMI was removed leaving the freshly transected axons at the distal nerve. To allow axonal fusion, the graft was excised in hypotonic saline containing a calcium chelator, as in previous protocols. Figure 4B: Intraoperative image at 20 weeks after graft showing the proximal common peroneal nerve pinned to nearby muscle and the TE-NMI pinned to the distal nerve with the intact tibial nerve running over it. Figure 4C: Intraoperative image immediately after non-immediate nerve repair showing the previously uninjured tibial nerve sutured to the distal portion of the common peroneal nerve after TE-NMI resection. The blue staining is from the application of methylene blue during the fusion protocol. Figure 4D: Compound nerve action potentials recorded immediately after non-instant nerve fusion were obtained in all animals that had undergone TE-NMI. Greater nerve conductance was observed in the TE-NMI group compared to the cell-free control. Figure 4E: Compound muscle action potentials were recorded after eliciting an evoked muscle response by stimulating proximal to the repair site. Greater evoked muscle responses were observed in the TE-NMI group compared to the cell-free control. Figure 4F: At 20 weeks post-repair, the surgical site was re-exposed and the TE-NMI grafts were harvested for histological analysis. Representative longitudinal images are shown in which neurons and dendrites were labeled with MAP2 (far-red) and sensory and motor TE-NMI neurons and axons were labeled with endogenous expression of GFP and tdTomato, respectively. Robust TE-NMI neuronal survival with axons spanning the lumen was seen at 20 weeks post-graft. Figure 4G: At higher magnification, healthy neurons were easily visualized within the micro-column co-labeled with MAP2. Figure 4H: Representative longitudinal nerve section and Figure 4I: Axonal nerve section just distal to the excised graft, labeled for Schwann cells (S100). Robust TE-NMI sensory outgrowth (GFP, green) was visualized. TE-NMI outgrowth was seen (TD-Tomato, red), but expression was weak. Error bars represent standard error.Means were compared using unpaired two-tailed Student's t-test. *p<0.05; **p<0.01. Scale bar: Fig. 4F 25 μm. [Diagram 5] 5A-5C: Electrophysiological functional recovery 1 month after non-immediate nerve repair. Figure 5A: Schematic illustrating electrophysiological outcome measures obtained at 1 month (24 weeks after initial nerve transection) after non-immediate nerve repair. Figure 5B: Compound nerve action potentials (CNAPs) were evoked in both groups, but larger responses and faster conduction velocities were observed in animals that had previously received a TE-NMI graft. Figure 5C: Compound muscle action potentials (CMAPs) were recorded in all animals with elevated evoked responses in the TE-NMI group. Mean values ​​were compared using unpaired two-tailed Student's t-test. Error bars represent standard error. *p<0.05; **p<0.01. [Figure 6]Figures 6A-6I show nerve morphometry and muscle reinnervation 1 month after non-immediate nerve repair. Figure 6A: Representative confocal images of nerve cross sections 5 mm distal to the repair site labeled for Schwann cells (S100), host / fused axons (SMI35), and myelin (myelin basic protein; mBP). Figure 6B: No numerical differences were observed in total axon numbers distal to the repair site. Figure 6C: An increase in host axon size was observed in animals that had previously received TE-NMI transplants. Figure 6D: By plotting the frequency distribution of axon diameters, a slight rightward shift could be noted in the TE-NMI group. Figures 6E and 6F: Representative confocal images of muscle cross sections of tibialis anterior (TA) muscles stained for acetylcholine receptors (bungarotoxin) and synaptophysin, a presynaptic marker, to identify the neuromuscular junction (NMJ). Figure 6G: No significant differences in total AchR counts between groups. Figure 6H: Greater muscle reinnervation was observed in animals that had previously received TE-NMI transplants, as indicated by the percentage of mature NMJs co-labeled for AchR and synaptophysin. Figure 6I: Increased muscle weight was observed in the TE-NMI group. Collectively, these findings suggest that while both groups demonstrate ongoing regeneration, TE-NMI may allow for more rapid axon maturation and muscle reinnervation after non-immediate nerve repair. Means were compared using unpaired two-tailed Student's t-tests. Error bars represent standard error. *p<0.05; **p<0.01. [Figure 7]Figures 7A-7C show mixed-modality TE-NMI neurite outgrowth comparison. Figure 7A: Representative confocal reconstructions at 3 DIV of mixed motor-sensory TE-NMI, consisting of populations of motor and sensory neurons plated at both ends. Motor and sensory neurons (DRG explants) were transduced to endogenously express GFP (green) or tdTomato (red), respectively. Figure 7B: Neurite outgrowth rates were calculated for motor axons extending into DRG explants (MN-DRG) and sensory axons extending into motor neurons (DRG-MN). Figure 7C: Representative confocal images at 14 DIV after immunocytochemistry to visualize motor neurons / axons (ChAT, far red) are shown. Error bars represent standard error. *p<0.05. Scale bar: 500 μm. [Figure 8]8A-8J show TE-NMI survival, growth, and integration with otherwise denervated nerves. FIG. 8A: Schematic illustrating the chronic host axotomy surgery model and experimental groups, including transplantation of an acellular column, one TE-NMI, or two TE-NMIs. Acellular controls were transplanted as negative controls. We hypothesized that TE-NMI would extend axons that interact with Schwann cells in otherwise denervated distal nerves. FIG. 8B: Representative images of microinjected TE-NMIs at 2 weeks post-transplantation, visualized following optical clearing and multiphoton microscopy. Robust TE-NMI neurons and axons (GFP) were found within the lumen protected from host cells invading the graft area. Figures 8C-8J: To assess whether TE-NMI axons extended and interacted with Schwann cells within otherwise denervated nerves, nerve cross sections taken 5 mm distal to the implantation site were labeled for TE-NMI axons (GFP), Schwann cells (S100β), nuclei (Hoechst; HST), and C-Jun (a gene encoding a pro-regeneration transcription factor found transiently in denervated Schwann cells). Figure 8C: High-resolution images showing examples of GFP+ TE-NMI axons extending through aligned Schwann cells resembling Büngner's zones. Figures 8D-8E: Greater growth was seen with GFP distal to two TE-NMIs than one TE-NMI, per nerve. Figure 8F: At higher magnification, Schwann cells were readily observed along with a subpopulation expressing C-Jun. Figure 8G: Many more cells were seen in the 2xTE-NMI cohort compared to the cell-free group. Figure 8H: Elevated C-Jun expression was also observed distal to the two TE-NMIs. Figure 8I: A larger number of Schwann cells (identified by colocalization of HST+ S100β) were also found in the 2×TE-NMI group. Figure 8J: A significant number of Schwann cells also colocalized with C-Jun distal to the two TE-NMIs. These findings suggest that TE-NMIs may interact with and potentially influence host S100+ Schwann cells in the denervated distal nerve at 6 weeks after transplantation in the host nerve axotomy model, potentially preserving a pro-regenerative phenotype as evidenced by C-Jun localization.Error bars represent standard error. Means were compared using one-way ANOVA followed by Tukey's post-hoc test. *p < 0.05. Scale bars: Fig. 8C: 100 μm, Fig. 8D: 100 μm, Fig. 8E: 5 μm. [Figure 9] Figures 9A-9H show nerve morphometry and muscle reinnervation 1 month after non-immediate nerve repair. Figure 9A: Representative confocal images of nerve cross sections 5 mm distal to the repair site were labeled for Schwann cells (S100), host axons (SMI35), and myelin (myelin basic protein; mBP). Figure 9B: No difference in SMI35 expression was detected distal to the repair site, suggesting that a comparable number of host axons regenerated within the distal sheath. Figure 9C: The average area of ​​SMI35+ regions seen distal to the repair was larger in the TE-NMI cohort, indicating that host axons in the distal nerve were larger than in the control group. Figure 9D: A greater number of myelinated axons were seen distal to the repair in the TE-NMI cohort. Figure 9E: Increased S100β expression, a common marker of Schwann cells, was observed in the TE-NMI group. Figure 9F: Representative confocal images (grayscale) of tibialis anterior (TA) muscle cross sections stained for acetylcholine receptors (bungarotoxin) to identify neuromuscular junctions (NMJs) and for the presynaptic marker synaptophysin. Sections were counterstained with phalloidin to visualize muscle fibers. Figure 9G: No significant difference in total AchR counts between groups. Figure 9H: Greater muscle reinnervation was seen in animals that had previously received TE-NMI transplants, as indicated by the percentage of mature NMJs co-labeled for AchR and synaptophysin. Taken together, these findings suggest that while both groups demonstrate ongoing regeneration, TE-NMI may enable even faster axon maturation and muscle reinnervation after non-immediate nerve repair. Partial areas were calculated by measuring the percent area of ​​positive fluorescent expression per ROI averaged across the three ROIs. Means were compared using an unpaired two-tailed Student's t-test. Error bars represent standard error. *p < 0.05; **p < 0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.Although any method and material similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.In describing and claiming the present invention, the following terminology is used:

[0008] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0009] As used herein, "about," when referring to a measurable value, e.g., amount, duration, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, where such variations are appropriate to perform the disclosed methods. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0010] As used in this specification and the claims, the terms "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them under U.S. patent law and can mean "includes," "including," and the like.

[0011] By "isolate" is meant obtaining one or more types of cells, purifying them to remove or substantially remove other cell types, and expanding them in primary culture.

[0012] As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, mammals such as livestock and pets, such as sheep, cows, pigs, dogs, cats and mice. Preferably, the subject is a human.

[0013] "Aggregates" and "neuronal aggregates" are used interchangeably to refer to aggregates or spheres of neurons and / or glial cells formed by centrifugation.

[0014] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0015] explanation Tissue engineered neuromuscular interfaces In one aspect, the present invention provides a tissue engineered neuromuscular interface, comprising: An extracellular matrix core, the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons extending at least in part along the extracellular matrix core; the population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core In various embodiments, the TE-NMI further comprises a hydrogel sheath coaxially surrounding the extracellular matrix core.

[0016] In various embodiments, the tissue engineered neuromuscular interface comprises: a second population of neurons at a second end of the extracellular matrix core, having axons extending at least in part along the extracellular matrix core; one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. A second population of said neurons Further includes:

[0017] In various embodiments, the population of neurons can be one or more neurons. In various embodiments, the population of neurons can be a neuronal aggregate. Neuronal aggregates are described in US Patent Application Publication No. 2019 / 0126043, which is incorporated herein by reference. Various methods for producing neuronal aggregates are known in the art. As a non-limiting example, the neuronal aggregate can be formed by centrifuging neurons in an inverted pyramidal well. In various embodiments, the neuronal aggregate can be a coaggregate comprising two or more types of neurons. The coaggregate can be formed by dissociating each type of neuron contained in the coaggregate and combining the dissociated neurons before forming the aggregate from the mixed neuronal population.

[0018] In various embodiments, the coaggregates have cross-sectional dimensions of about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 700 μm, about 700 μm to about 1000 μm, about 1000 μm to about 1500 μm, about 1500 μm to about 2000 μm, and about 2500 μm to about 3000 μm.

[0019] In various embodiments, the extracellular matrix core has a maximum cross-sectional dimension selected from the group consisting of about 10 μm to about 25 μm, about 25 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 700 μm, and about 700 μm to about 1000 μm, about 1000 μm to about 1500 μm, and about 1500 μm to about 2000 μm, and about 2000 μm to about 2500 μm, and about 2500 μm to about 3000 μm.

[0020] In various embodiments, the hydrogel sheath may be from about 20 μm to about 50 μm, from about 50 μm to about 100 μm, from about 100 μm to about 200 μm, from about 200 μm to about 250 μm, from about 250 μm to about 300 μm, from about 300 μm to about 350 μm, from about 350 μm to about 400 μm, from about 400 μm to about 450 μm, from about 450 μm to about 50 In various embodiments, the hydrogel sheath has a maximum cross-sectional dimension selected from the group consisting of about 701 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, about 800 μm to about 1200 μm, about 1200 μm to about 1700 μm, and about 1700 μm to about 2200 μm, and about 2200 μm to about 2700 μm, and about 2700 μm to about 3200 μm. In various embodiments, the hydrogel sheath has a maximum cross-sectional dimension of about 701 μm and the extracellular matrix core has a maximum cross-sectional dimension of about 300 μm.

[0021] In various embodiments, the tissue engineered neuromuscular interface has a length of about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, about 800 μm to about 1200 μm, about 1200 μm to about 1500 μm, and about 1500 μm to about 2000 μm.

[0022] In various embodiments, the tissue engineered neuromuscular interface further comprises one or more non-neuronal cells selected from the group consisting of endothelial cells, myocytes, myoblasts, astrocytes, olfactory ensheathing cells, oligodendrocytes, or Schwann cells.

[0023] In various embodiments, the neuron is derived from stem cells or isolated from dorsal root ganglion.In various embodiments, the neuron is heterologous neuron, autologous / patient-specific neuron, allogeneic neuron, whole dorsal root ganglion or sensory explant.In various embodiments, the neuron is heterologous neuron derived from wild type or transgenic pig.

[0024] In various embodiments, the extracellular matrix core comprises collagen, gelatin, laminin, fibrin, fibronectin and / or hyaluronic acid, hi various embodiments, the hydrogel sheath comprises agarose, collagen, gelatin, silk, chitosan, fibrin and / or hyaluronic acid.

[0025] A method to preserve the regenerative potential of distal nerve segments by implanting a tissue engineered neuromuscular interface In another aspect, the present invention provides a method of preserving the regenerative capacity of a distal nerve segment following peripheral nerve injury in a subject in need thereof, comprising the steps of: Implanting one or more tissue engineered neuromuscular interfaces (TE-NMIs) at a distal site of a distal nerve segment, The TE-NMI: An extracellular matrix core, the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons extending at least in part along the extracellular matrix core; the population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core The implanting step comprises: In various embodiments, the TE-NMI further comprises a hydrogel sheath coaxially surrounding the extracellular matrix core.

[0026] In various embodiments, the TE-NMI comprises: a second population of neurons at a second end of the extracellular matrix core, having axons extending at least in part along the extracellular matrix core; one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. A second population of said neurons Further includes:

[0027] Without meaning to be limited by theory, in various aspects and embodiments, and as further discussed in the Examples herein, transplantation of one or more TE-NMIs into a distal segment of an injured peripheral nerve allows neurons within the TE-NMI to grow axons to the distal nerve segment, which preserve the regenerative capacity of the distal nerve segment that would otherwise be lost.

[0028] In various embodiments, the transplantation is performed immediately after injury. In various embodiments, the injury results from surgery. In various embodiments, the transplantation is performed less than 24 hours after injury. In various embodiments, the transplantation is performed less than 7 days after injury. In various embodiments, the transplantation is performed less than 2 weeks after injury. In various embodiments, the transplantation is performed less than one month after injury. In various embodiments, the transplantation is performed one month or more after injury.

[0029] In various embodiments, one or more TE-NMIs are implanted end-to-side, intrafascicularly, or serially at the distal nerve segment. In various embodiments, implantation of one or more TE-NMIs is ultrasound-guided or MRI-guided. In various embodiments, at least two tissue-engineered neuromuscular interfaces are implanted at the distal nerve segment. In various embodiments, at least five tissue-engineered neuromuscular interfaces are implanted at the distal nerve segment. In various embodiments, at least ten tissue-engineered neuromuscular interfaces are implanted at the distal nerve segment.

[0030] In various embodiments, the method further comprises performing a primary nerve repair procedure to treat the peripheral nerve injury, hi various embodiments, the primary nerve repair procedure comprises direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or tissue engineered nerve graft.

[0031] Methods for treating peripheral nerve injury by implanting a tissue engineered neuromuscular interface In another aspect, the present invention provides a method of treating peripheral nerve injury in a subject in need thereof, comprising the steps of: Implanting one or more tissue engineered neuromuscular interfaces (TE-NMIs) at a distal site of a distal nerve segment, The TE-NMI: An extracellular matrix core, the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons extending at least in part along the extracellular matrix core; the population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core said implanting step comprising: monitoring extrinsic axonal growth throughout the otherwise denervated distal segment for innervation of muscles and / or sensory end organs; removing one or more tissue engineered neuromuscular interfaces at the distal nerve segment; and performing a primary nerve repair procedure, thereby treating a peripheral nerve injury; In various embodiments, the TE-NMI further comprises a hydrogel sheath coaxially surrounding the extracellular matrix core.

[0032] In various embodiments, the TE-NMI comprises: a second population of neurons at a second end of the extracellular matrix core, having axons extending at least in part along the extracellular matrix core; one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. A second population of said neurons Further includes:

[0033] Without meaning to be limited by theory, implantation of TE-NMI contributes to improved efficacy of primary nerve repair procedures by preserving the regenerative capacity of the distal nerve segment. Exogenous axons promote Schwann cell expression in the distal nerve and integrate with otherwise degenerating muscle and / or sensory terminal targets, increasing the upper limit of functional recovery after non-immediate nerve repair.

[0034] In various embodiments, the primary nerve treatment includes direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or tissue engineered nerve graft implantation. In various embodiments, the TE-NMI is removed less than one week after implantation. In various embodiments, the TE-NMI is removed less than one month after implantation. In various embodiments, the TE-NMI is removed less than one year after implantation. In various embodiments, the TE-NMI is removed one year or more after implantation.

[0035] Methods for treating peripheral nerve injuries by implanting a tissue engineered neuromuscular interface and fusing it with the proximal nerve In another aspect, the present invention provides a method of treating peripheral nerve injury in a subject in need thereof, comprising the steps of: Implanting one or more tissue engineered neuromuscular interfaces (TE-NMIs) at a distal site of a distal nerve segment, The TE-NMI: An extracellular matrix core, the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons extending at least in part along the extracellular matrix core; the population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core said implanting step comprising: monitoring extrinsic axonal growth throughout the otherwise denervated distal segment for innervation of muscles and / or sensory end organs; removing one or more tissue engineered neuromuscular interfaces at the distal nerve segment; and Fusing the TE-NMI axons in the distal nerve segment with at least one proximal axon. In various embodiments, the TE-NMI further comprises a hydrogel sheath coaxially surrounding the extracellular matrix core.

[0036] In various embodiments, the TE-NMI comprises: a second population of neurons at a second end of the extracellular matrix core, having axons extending at least in part along the extracellular matrix core; one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. A second population of said neurons Further includes:

[0037] In various embodiments, primary nerve repair is performed. In various embodiments, the primary nerve treatment includes direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or tissue engineered nerve graft implantation. In various embodiments, a free radical scavenger is applied prior to primary nerve repair. In various embodiments, the free radical scavenger is methylene blue.

[0038] In various embodiments, the axons extending from the TE-NMI are transected when the TE-NMI is removed and fused with the proximal nerve segment. Nerve fusion using an extension-grown tissue-engineered nerve graft is described in US Patent Application Publication No. 2020 / 0230293, which is incorporated herein by reference. Various methods for fusing neurons are known in the art, and the skilled artisan will select an appropriate method. In various embodiments, a reagent is applied before removing the TE-NMI to inhibit axonal degeneration. In various embodiments, the reagent comprises hypotonic saline or a calcium chelator. In various embodiments, the reagent is a hypotonic saline containing a calcium chelator. In various embodiments, the exogenous neurons are genetically modified, such as SARM1 knockdown, to inhibit Wallerian degeneration.

[0039] In various embodiments, a fusogenic agent is applied during primary nerve repair to promote membrane sealing. In various embodiments, the fusogenic agent is polyethylene glycol or chitosan. In various embodiments, application of the fusogenic agent promotes nerve regeneration and functional recovery. EXAMPLES

[0040] Experimental Example The present invention will be described in more detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0041] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Therefore, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0042] Example 1: material and method The materials and methods used to carry out the following examples are now described.

[0043] Isolation of embryonic neurons and aggregation of spinal cord motor neurons Spinal cords and dorsal root ganglia (DRGs) were isolated from embryonic day 16 Sprague-Dawley rats (Charles River, Wilmington, mA) as previously described. DRG explants were kept overnight in Hibernate-E and transduced with AAV2 / 1.hSynapsin.EGFP.WPRE.bGH (UPenn Vector Core). Motor neuron aggregates were formed following force-induced aggregation of spinal motor neurons isolated from dissociated spinal cords using Optiprep density gradients as previously described. Motor neuron aggregates were incubated overnight in culture medium and transduced with AAV.hSynapsin.tdTomato vector. Neurons were plated in spinal cord astrocyte-conditioned neurobasal medium + 10% FBS supplemented with 37 ng / mL hydrocortisone, 2.2 μg / mL isobutylmethylxanthine, 10 ng / mL BDNF, 10 ng / mL CNTF, 10 ng / mL CT-1, 10 ng / mL GDNF, 2% B-27, 20 ng / mL NGF, 20 μM mitotic inhibitors, 2 mM L-glutamine, 417 ng / mL forskolin, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 2.5 g / L glucose.30.

[0044] Fabrication of microcolumns Agarose or agarose-gelatin hydrogel microcolumns were constructed using a three-phase process similar to that described previously. Briefly, agarose microcolumns were formed using glass capillaries (345–701 μm) to allow the insertion of an acupuncture needle (180–350 μm) through the lumen. Molten agarose (3% weight / volume) in Dulbecco's phosphate-buffered saline (DPBS) was added to the capillary containing the acupuncture needle and allowed to cool. The acupuncture needle was quickly removed to create a hydrogel shell, and the microcolumn was stored in DPBS at 4 °C. Agarose-gelatin microcolumns (1.5% agarose + 1.5% gelatin) were fabricated as described above, except that the microcolumns were stored in 100 μL in 7 mL of DPBS overnight at room temperature, and then washed three times in DPBS before further experiments. All microcolumns were cut to appropriate lengths, UV sterilized for 30 min, and stored in DPBS at 4 °C.

[0045] The microcolumns were transferred to a new Petri dish, excess DPBS was removed from the lumen of the microcolumn via a micropipette, and replaced with extracellular matrix (ECM) composed of 1.0 mg / ml rat tail collagen + 1.0 mg / ml mouse laminin (Reagent Proteins, San Diego, CA). DRG explants or motor neuron aggregates were carefully placed at the end of the microcolumn containing the ECM under stereoscopic magnification using fine forceps and incubated at 37°C, 5% CO 2 The microcolumns were then allowed to adhere for 45 min at RT. Sensory TE-NMI were made by seeding DRG explants on both ends of the microcolumns. Motor TE-NMI were made by seeding motor neuron aggregates on both ends of the microcolumns. Mixed motor and sensory TE-NMI were made by seeding motor neuron aggregates and DRG explants on opposite ends of the microcolumns. TE-NMI were then returned to culture and grown with fresh medium changes every other day.

[0046] For characterization of mixed TE-NMIs, motor neurons were transduced overnight to endogenously express GFP and sensory neurons were transduced overnight to endogenously express tdTomato prior to plating. For the remaining in vitro characterization and in vivo performance, motor neurons were transduced overnight with tdTomato and sensory neurons were transduced overnight with GFP. All TE-NMIs were returned to culture after creation with half the medium changed every other day.

[0047] immunocytochemistry TE-NMI were fixed in 4% paraformaldehyde for 35 min, rinsed in 1x PBS, permeabilized with 0.3% Triton X100 + 4% horse serum in PBS for 60 min, and then incubated with primary antibodies overnight at 4°C. Primary antibodies were Tuj-1 / beta-III tubulin (T8578, 1:500, Sigma-Aldrich) to label axons, and synapsin-1 (A6442, 1:500, Invitrogen) to label presynaptic specializations. After primary antibody incubation, TE-NMI were rinsed in PBS and incubated with fluorescently tagged secondary antibodies (1:500; Invitrogen) for 2 h at 18°C–24°C. Finally, Hoechst (33342, 1:10,000, ThermoFisher) was added for 10 min at 18°C–24°C, and then rinsed in PBS. TE-NMIs were imaged with a Nikon A1RSI laser scanning confocal microscope coupled with NIS Elements AR 4.50.00. For each fluorescent channel, consecutive slices of 10-20 μm were acquired in the z-plane. All confocal images shown are maximum intensity projections of confocal z-slices.

[0048] In vitro TE-NMI imaging Phase contrast microscopy images of TE-NMI were taken over days in vitro (DIV), neurite length was measured, and growth rate was calculated. TE-NMI viability and the presence of the desired neuronal phenotype were quantified using a Nikon Eclipse Ti-S microscope coupled with a QIClick camera and NIS Elements BR 4.13.00 at 10x magnification.

[0049] Confocal imaging of TE-NMI was performed on a Nikon A1RSI laser scanning confocal microscope coupled with NIS Elements AR 4.50.00. For each fluorescent channel, consecutive slices of 10-20 μm were acquired in the z-plane. All confocal images shown are maximum intensity projections of confocal z-slices.

[0050] Chronic Rodent Sciatic Nerve Axotomy and Acute TE-NMI Implantation The ability of TE-NMI to integrate with denervated distal nerves was evaluated in a rodent model of chronic sciatic nerve axotomy. Sprague-Dawley rats were anesthetized with isoflurane and the hind limbs were scrubbed with betadine. Meloxicam (2 mg / kg) was administered subcutaneously in the nape of the neck and bupivacaine (2 mg / kg) was administered subcutaneously along the incision. The gluteal muscles were dissected to expose the sciatic nerve exiting the sciatic notch.

[0051] TE-NMI (3 mm long) were implanted into the distal nerve using three different surgical paradigms. In a proof-of-concept experiment, intraneural TE-NMI implantation was performed in some animals to demonstrate survival of TE-NMI after microinjection. Briefly, the sciatic nerve was exposed as described above. TE-NMI was loaded into a Hamilton syringe and deposited intraneurally. The epineurium of the sciatic nerve was carefully dissected, and the needle containing TE-NMI was inserted into the exposed fiber bundle and advanced 7 mm into the nerve, TE-NMI was deposited intraneurally, and the epineurium was closed with 8-0 prolene. The nerve was sharply transected, and the proximal stump was inserted into nearby muscle. Viability of TE-NMI was assessed 2 weeks after implantation using tissue clearing and multiphoton microscopy.

[0052] To assess sensory TE-NMI outgrowth and Schwann cell coverage within the denervated distal nerve, a 5 mm segment of the sciatic nerve was resected 5 mm proximal to the trifurcation and the proximal nerve was covered with Teflon tape or fixed to nearby muscle. The sensory TE-NMI was placed within a 5 mm nerve wrap (Stryker Orthopedics, Kalamazoo MI) that was fixed to the nerve to provide a protective environment for the nerve and TE-NMI. Approximately 100 μl of 2 mg / ml collagen ECM was applied within the wrap to promote outgrowth of the TE-NMI axons in the distal nerve. Animals were randomly assigned to the following groups: (A) one sensory TE-NMI (n=5); (B) two sensory TE-NMI (n=5), (C) acellular control microcolumns containing ECM only (n=5).

[0053] To assess chronic integration with denervated muscles, mixed motor-sensory TE-NMI were implanted within a nerve wrap as described above. In this experiment, the common peroneal nerve was resected to its proximal origin and the nerve was transected 5 mm distal to the bifurcation. The proximal common peroneal nerve stump was inserted into a nearby muscle. The mixed motor-sensory TE-NMI was placed in a 5 mm nerve wrap (Stryker Orthopedics, Kalamazoo MI) that was secured to the nerve to provide a protective environment for the nerve and TE-NMI. Approximately 100 μl of 2 mg / ml collagen ECM was applied within the wrap to promote outgrowth of TE-NMI axons in the distal nerve. Animals were randomly assigned to the following groups: (A) TE-NMI (n=5); (B) microcolumn control (n=5), (C) no implant control (n=5).

[0054] In all procedures, the surgical site was closed with 4-0 absorbable Vicryl sutures and skin staples. Animals were harvested and returned to the animal facility for the duration of the study.

[0055] Non-instant axonal fusion At 20 weeks after grafting and chronic axotomy, animals were anesthetized and the surgical site was re-exposed. The surgical site was perfused with calcium-free PlasmaLyte-A with calcium chelator, the graft was isolated, and the distal common peroneal nerve was sharply transected. The tibial nerve was sharply transected while bathing the transected nerve with additional PlasmaLyte-A, and the proximal tibial nerve and distal common peroneal nerve were secured using two 8-0 prolene sutures to complete the nerve repair with standard end-to-end sutures. Just before tightening the sutures, a hypotonic 1% methylene blue solution was applied to the nerve terminals, followed by administration of high molecular weight polyethylene glycol (3350 MW). Calcium-containing lactated Ringer's solution was applied to the wound to wash out excess PEG. Electrophysiological recordings were performed immediately before and after repair to assess acute functional recovery, as described below. The deep layers and skin were closed, and the area was covered as above.

[0056] immunohistochemistry At the end of the day, animals were euthanized by intracardiac injection of Euthasol. Nerves were removed and then fixed in formalin at 4°C for 24 hours, then rinsed in PBS for another 24 hours. Muscles were removed and placed in paraformaldehyde at 4°C for 24 hours, then cryoprotected in 20% sucrose.

[0057] For histological evaluation after transplantation, tissues were placed in 30% sucrose overnight, embedded in optimal cutting medium, and then frozen in dry ice / isopentane. The transplantation site was sectioned longitudinally, and the area 5 mm distal to the graft was sectioned axially at a thickness of 20 μm and mounted on glass slides for staining. Frozen sections were washed three times in PBS, blocked, and permeabilized in 4% normal horse serum with 0.3% Triton X-100 for 1 h. All subsequent steps were performed with blocking solution for antibody dilution. Neurons were labeled with chicken anti-MAP2 (1:500, Abcam, ab532) and Schwann cells were labeled with anti-S100 (1:500, Invitrogen, PA1-38585). Primary antibodies were applied overnight at 4°C, followed by the appropriate fluorochrome-conjugated secondary antibodies (1:1000; AlexaFluor, Invitrogen) for 2 h at room temperature. Hoechst was applied (1:10,000) before mounting and coverslipping with Fluoromount G. Sensory neurons / axons were visualized by endogenous GFP expression and motor neurons by endogenous tdTomato expression.

[0058] For cross-sectional histological evaluation of the distal nerve after non-immediate nerve repair, a 1 cm nerve segment distal to the repair zone was embedded in paraffin. The blocks were then mounted on a microtome, sectioned axially at 8 μm thickness, mounted on glass slides, and prepared for staining as follows: Axial sections were deparaffinized in xylene and rehydrated in a descending gradient of ethanol. After rehydration, antigen retrieval was performed in TRIS / EDTA buffer for 8 min using a modified pressure cooker / microwave method. Normal horse serum in Optimax (Biogenex) was then applied to the sections (VectaStain Universal kit, according to the manufacturer's instructions). Sections were incubated with mouse anti-SMI35 (1:1000, Covance, SMI-35R), rabbit anti-S100 (1:500, Invitrogen, PA1-38585), and chicken anti-myelin basic protein (Encor, CPCA-MBP; 1:1500) in Optimax + normal horse serum overnight at 4°C (VectaStain Universal kit, according to manufacturer's instructions). Sections were washed three times for 5 min with PBS / TWEEN before application of the appropriate fluorochrome-conjugated secondary antibodies (1:1000; AlexaFluor, Invitrogen) for 1 h at room temperature. After rinsing three times for 5 min with PBS / TWEEN, the antibodies were applied for 20 min. Finally, sections were washed and coverslipped as above.

[0059] For muscle cross-sectional histological analysis, tibialis anterior muscles were harvested and stored overnight in 2% paraformaldehyde. Muscles were cryoprotected overnight in 20% sucrose, blocked, frozen, sectioned axially at 20 μm thickness, and stained according to the protocol described above. To identify muscle actin, sections were incubated with AlexaFluor488-conjugated phalloidin (1:400, Invitrogen, A12379) for 2 h at room temperature. Adjacent sections were incubated overnight at 4°C with rabbit anti-synaptophysin (1:500, abcam, ab32127) to identify presynaptic vesicles, followed by simultaneous application of AlexaFluor-568 antibody (1:500, ThermoFisher, A10042) and AlexaFluor-647-conjugated bungarotoxin for 2 h at room temperature to identify postsynaptic receptors (1:1000, Invitrogen, B35450).

[0060] Tissue clearing Some nerves were excised for tissue clearing using the Visikol protocol. Briefly, after fixation in formalin at 4°C for 24 hours, the nerves were rinsed with PBS overnight at 4°C and dehydrated in a series of ethanol washes (30%, 50%, 70% and 90%) for 2 hours each and 100% ethanol for 24 hours. The nerves were then incubated in Visikol 1 for 24 hours followed by Visikol 2 for at least 24 hours to complete the clearing process. Multiphoton microscopy (Nikon) was used to visualize TE-NMI viability within the graft area.

[0061] Functional evaluation At 16 weeks after axotomy, compound muscle action potentials (CMAPs) were assessed to evaluate evoked muscle responses. Animals were re-anesthetized and a bipolar subcutaneous stimulating electrode was placed superficial to the common peroneal nerve. A monopolar subcutaneous recording electrode was placed on the tibialis anterior muscle and a reference electrode was placed on its tendon. A handheld bipolar hook electrode (Rochester Electro-Medical, Lutz, FL; #400900) was used to stimulate the nerve (biphasic; amplitude: 0–10 mA; duration: 0.2 ms; frequency: 1 Hz). Supramaximal CMAP recordings were acquired and averaged over a series of 5 pulses (100x gain; 10–10,000 Hz bandpass and 60 Hz notch filter; Natus Viking EDX). At 20 weeks after axotomy, animals were re-anesthetized and the surgical site was exposed. CMAPs were recorded by stimulating the distal nerve prior to non-immediate nerve repair. After non-immediate nerve repair, proximal and distal CMAPs were recorded by stimulating 5 mm proximal or distal to the repair site, respectively. The mean peak-to-baseline amplitude was recorded.

[0062] To assess immediate electrical conduction across the repair site, compound nerve action potentials (CNAPs) were recorded by stimulating the proximal stump with a bipolar hook electrode and recording with a bipolar hook electrode (1000x gain; 10–10,000 Hz bandpass and 60 Hz notch filter; Natus Viking EDX). The mean peak-to-peak amplitude was recorded and conduction velocity was calculated by dividing the distance between the electrodes by the latency.

[0063] One month after non-immediate nerve repair (a total of 24 weeks after chronic host axotomy), CNAP and CMAP recordings were obtained as described above.

[0064] Data acquisition and statistical analysis Neuronal constructs were imaged using phase contrast or epifluorescence microscopy on a Nikon Eclipse Ti-S with digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01). Fluorescence images were acquired on a Nikon A1R confocal microscope (1024 × 1024 pixels) with a 10× air objective and a 60× oil objective using Nikon NIS-Elements AR 3.1.0 (Nikon Instruments, Tokyo, Japan). Multiple confocal z-stacks were digitally captured and analyzed, and all were tiled and reconstructed across the entire section and z-stack thickness.

[0065] For all TE-NMI neurite outgrowth assays, the longest neurite was measured from the edge of the aggregate (n ≥ 4–6 TE-NMI per condition per time point). For characterization of TE-NMI fabrication, mean neurite outgrowth was compared by repeated measures two-way analysis of variance (ANOVA) with cell type and biomaterial hydrogel containment as the two independent variables at 1 and 3 DIV.

[0066] All histological evaluations were performed at the implantation site (longitudinal freeze), 5 mm distal to the implantation site (axial freeze), or 5 mm distal to the non-immediate nerve repair (axial paraffin). For frozen tissue, TE-NMI neurons / axons were identified as SMI35 negative and GFP positive for sensory neurons / axons, or tdTomato positive for motor neurons / axons. For paraffin tissue, SMI35 only labeled host regenerating / fused axons.

[0067] For quantification of all axonal morphometry, measurements were calculated from confocal z-stack maximum projections and analyzed using FIJI software. An automated image processing macro was used to minimize potential bias. Individual channels were separated using MaxEntropy thresholding and then quantified using the "Analyze Particles" function for features with an area >1 µm2 to minimize noisy signals. Total number of segmented particles, size of segmented particles, and percent area covered were calculated from 2–3 sections per animal. Mean values ​​were obtained by averaging values ​​per animal across groups for further statistical analysis.

[0068] For TE-NMI outgrowth and host Schwann cell (S100) reactivity at 6 weeks after graft / host axotomy, mean values ​​were compared by one-way analysis of variance (ANOVA) between the following groups: (a) one TE-NMI, (b) two TE-NMI, (c) micro-column only. For evoked muscle responses at 16 weeks after graft / host axotomy, mean CMAP amplitudes were compared by one-way ANOVA between the following groups: (a) TE-NMI, (b) micro-column only, and (c) injury only / no graft.

[0069] To quantify the total number of acetylcholine receptors (AchRs) and the percentage of mature neuromuscular junctions, each muscle section was first imaged at low magnification by an investigator blinded to the experimental groups to identify bungarotoxin (BGX)-positive cluster regions of AchRs (10x air objective, 1024 x 1024). Next, three regions of interest (ROIs) were randomly selected and automatically acquired (2x2 regions, 60x oil objective with 2x digital zoom, 2048 x 2048) without the investigator visualizing synaptophysin channels prior to acquisition. The total number of BGX-positive cells or AchR receptors was quantified from the low magnification images for each animal. Mature neuromuscular junctions (NMJs) were identified as BGX-positive cells co-labeled with synaptophysin. The mean percent of mature NMJs was calculated by dividing the number of mature NMJs by the total number of bungarotoxin-positive receptors, averaged across replicates and by group.

[0070] For all statistical analyses after non-immediate nerve repair, these samples were analyzed as a single group (no cells) since no differences were detected between the negative control groups (micro-column only and injury only / no implantation). Electrical conduction and evoked muscle responses were performed immediately after non-immediate nerve repair (20 weeks after initial host axotomy) and 1 month after non-immediate nerve repair (24 weeks after initial host axotomy) by comparing the mean CMAP amplitude, CNAP amplitude and CNAP velocity using unpaired two-tailed Student's t-tests. Mean host axon number and size, AChR number, and percent mature NMJs were compared at 1 month after non-immediate nerve repair (24 weeks after initial host axotomy) using unpaired two-tailed Student's t-tests.

[0071] Post-hoc Tukey's pairwise comparisons were performed when differences existed between groups following one-way ANOVA. All statistical tests required p < 0.05 for significance and were performed in GraphPad Prism 9 for Windows 64-bit (La Jolla California USA). Mean values ​​are presented as mean ± SEM unless otherwise noted.

[0072] result Tissue engineered neuromuscular interfaces (TE-NMIs) are preformed, axon-rich microtissues within a protective biomaterial TE-NMI is an anatomically inspired neural construct composed of a discrete population of neurons spanning long axonal tracts similar to the neuron-axon tissue of the nervous system (Figure 1). We engineered microtissues composed of sensory neuron-axons only (sensory TE-NMI), motor neuron-axons only (motor TE-NMI), or a mixed population of motor neuron-axons and sensory neuron-axons (mixed TE-NMI) (Figure 1A). In initial experiments, robust sensory axon growth was observed across dorsal root ganglia (DRG) spaced 5 mm apart within a large macroscale agarose column with an outer diameter of 2 mm and an inner diameter of 1 mm that contained collagen extracellular matrix (ECM) (Figure 1B). Next, the microtissue engineering fabrication technique was adapted from methodologies previously developed in our laboratory for Parkinson's disease, spinal cord injury, and brain-machine interfacing. The smallest TE-NMI was 3 mm long with an outer diameter of 350 μm and an inner diameter of 180 μm (Figure 1C); however, because our goal was to match the nerve bundle structure within the nerve, the TE-NMIs in the following studies were 3-5 mm long with an outer diameter of 701 μm and an inner diameter of 300 μm unless otherwise noted.

[0073] For motor TE-NMI and mixed TE-NMI, aggregated embryonic spinal cord motor populations were formed and subsequently plated on the ends of microcolumns as previously described. Healthy neuronal and neurite outgrowth was observed by phase contrast microscopy. TE-NMI immunocytochemistry confirmed the motor neuron phenotype by co-labeling for the neuronal / axonal markers Tuj1 and ChAT (Figure 1D). Although agarose is a relatively inert biomaterial, it has a long degradation time into non-absorbable by-products that may hinder translation. Therefore, an alternative biocontainer consisting of an agarose-gelatin composite hydrogel was evaluated (Figure 1E). At 1 day in vitro (DIV), sensory outgrowth in agarose-gelatin microcolumns was faster than motile outgrowth in either agarose or agarose-gelatin microcolumns. However, by 3 DIV, sensory outgrowth in the agarose-gelatin microcolumns was greater than in the other groups, whereas motor outgrowth was enhanced in the agarose microcolumns compared to the agarose-gelatin microcolumns (Figure 1F). These findings support previous studies showing that sensory axons extending from DRG explants are often faster than motor neurons. Interestingly, these data suggest that motor neurons preferentially grow in the agarose microcolumns despite the presence of active moieties in the agarose-gelatin microcolumns. To assess preferential outgrowth from motor or sensory neurons, neurite outgrowth was measured in mixed TE-NMI. Faster motor axonal outgrowth extending toward sensory neurons was seen at 3 DIV (Figures 7A-7C). Based on these findings, agarose microcolumns were chosen for in vivo experiments to improve the probability of successful motor outgrowth and to provide more protection from the host immune response after transplantation due to its slow degradation rate.

[0074] TE-NMI preserves Schwann cells in otherwise denervated nerves 6 weeks after transplantation. To assess whether TE-NMI could preserve the regenerative capacity of the distal nerve, the sciatic nerve was transected, TE-NMI was attached to the distal nerve, and the proximal stump was capped to prevent host regeneration (Figure 2A). In a proof-of-concept experiment, TE-NMI was microinjected into the denervated distal nerve by "laying out" the construct (Figure 2B). At 2 weeks, optical clearing and two-photon microscopy found robust, transplanted TE-NMI neurons and axons within the lumen protected by the outer containment (Figure 2C).

[0075] To test whether TE-NMI preserve Schwann cell expression, we used a model of chronic nerve axotomy (Figure 2A). In this study, one or two TE-NMI were transplanted into a conduit with a fixed distal sciatic stump. We hypothesized that early reinnervation of otherwise denervated Schwann cells with TE-NMI axons would preserve Schwann cell expression after long-term host axotomy. Indeed, 6 weeks after transplantation, robust sensory TE-NMI axonal growth was observed, extending at least 5 mm into the host tissue (Figure 2D). Of note, host S100+ Schwann cells were closely aligned with GFP+ axons, and in some cases, GFP+ axons were visualized extending through Schwann cells resembling Wüngner's zone (Figure 2E). After implantation of two TE-NMIs, we observed greater GFP+ axonal growth (Figure 2F) and Schwann cell coverage (Figure 2G) in the otherwise denervated nerves, whereas reduced Schwann cell expression was observed in the other groups. These findings suggest that exogenous axons extending from the TE-NMIs may maintain a pro-regenerative environment necessary for regenerating host axons to reinnervate distal targets after repair.

[0076] S100β+ Schwann cells and c-Jun expression were also quantified 6 weeks after transplantation using automated segmentation in relation to total cell number based on nuclear (Hoechst+) staining (Figure 8F). The total number of Hoechst+ cells was higher in the 2×TE-NMI cohort (303.3 ± 49.35 cells / 40,000 μm2) than in the acellular group (202.2 ± 41.36 cells / 40,000 μm2; F(2, 9) = 4.44; p = 0.0376; Figure 8G). Furthermore, among Hoechst+ cells, there was higher co-expression of S100β in the 2×TE-NMI group (249.1 ± 30.29 cells / 40,000 μm2) than in the 1×TE-NMI (155.2 ± 17.91 cells / 40,000 μm2; p = 0.0038) or cell-free cohorts (141.2 ± 10.22 cells; F(2, 9) = 15.78; p = 0.0015; Figure 8I). Similarly, among Hoechst- and S100β+ cells, we observed greater colocalization with c-Jun in the 2×TE-NMI cohort (249.1 ± 15.14 cells / 40,000 μm2) than in the 1×TE-NMI (155.2 ± 35.82 cells / 40,000 μm2; p = 0.0095) or cell-free groups (141.2 ± 20.44 cells / 40,000 μm2; F(2, 9) = 9.883; p = 0.0102; Figure 2J). Interestingly, we also found increased C-Jun expression in the 2xTE-NMI cohort (517.5 ± 71.06 cells / 40,000 μm2) compared to the 1xTE-NMI (225.1 ± 23.09 cells / 40,000 μm2; p = 0.0003) and acellular groups (239.1 ± 83.77 cells / 40,000 μm2; F(2, 9) = 25.91; p = 0.0005; Figure 8H). These findings suggest that exogenous axons extending from TE-NMI may maintain a pro-regenerative environment necessary for regenerating host axons to reinnervate distal targets after repair.

[0077] TE-NMI maintains electrophysiological activity of distal muscle targets 16 weeks after implantation Next, we investigated whether a mixed sensory-motor TE-NMI could preserve otherwise denervated muscles (Figures 3A-3F). The distal common peroneal nerve was chosen for the surgical paradigm because its single-bundle nerve structure more closely matches the TE-NMI size in size, avoiding the need for multiple constructs. After fixing the TE-NMI to the otherwise denervated distal common peroneal nerve, the severed proximal nerve stump was attached to the adjacent muscle to prevent host regeneration (Figure 3A). We chose a mixed motor-sensory TE-NMI (Figure 3B) because sensory axons are effective in preserving Schwann cells, and we hypothesized that the addition of motor axons would be beneficial in preserving muscle electrophysiological responses after chronic host axotomy. However, based on our previous data, motor neurons co-cultured with sensory neurons appear to grow better in vitro, improving nerve regeneration and increasing muscle reinnervation, allowing for greater functional recovery (Figures 7A-7C).

[0078] To test whether the mixed TE-NMI integrated with the otherwise denervated distal muscle targets, transcutaneous stimulation was performed 16 weeks after implantation. Greater evoked muscle responses were observed in the mixed TE-NMI group compared to the no implant or microcolumn only controls (Figures 3C, 3D). These findings indicate that despite the lack of host axons due to the proximal nerve cap preventing host regeneration, TE-NMI containing motor and sensory neurons project axons and functionally integrate with otherwise denervated muscles, allowing for greater electrophysiological muscle responses.

[0079] TE-NMI provides exogenous axons in otherwise denervated distal nerve sheaths, allowing non-instant axonal fusion. Based on these findings, we hypothesized that TE-NMI axons, which extend within otherwise denervated nerves and subsequently integrate with muscles, would be amenable to axonal fusion following standard PEG fusion protocols. At 20 weeks after TE-NMI implantation, the distal nerve was freshly axotomized for nerve fusion by resecting the TE-NMI (Figure 4B). To test whether TE-NMI implantation allowed delayed fusion and promoted functional recovery, we utilized a cross-suture repair model to avoid the need for grafting between retracted proximal and distal stumps and minimize disruptions associated with long-term proximal neuronal injury. Non-immediate cross-suture repair was completed by fixing the proximal stump of the previously uninjured tibial nerve to the distal end of the freshly axotomized common peroneal nerve containing the TE-NMI axons (Figures 4A, 4C).

[0080] In all TE-NMI animals, immediate electrical conduction was obtained after non-immediate nerve fusion compared to none in any of the control groups (Figure 4D). Similarly, greater evoked muscle responses were recorded in the TE-NMI cohort after proximal stimulation (Figure 4E). Within the harvested graft sites, robust TE-NMI neuronal survival was visualized within the micro-columns at 20 weeks post-repair (Figure 4F, 4G). Although no host axons were visualized immediately distal to the resected grafts, sensory and motor axons transduced prior to transplantation were observed extending from the TE-NMI into the host distal nerve (Figure 4H, 4I).

[0081] TE-NMI allows greater electrophysiological recovery, axonal maturation, and muscle reinnervation after non-immediate nerve repair At 1 month after non-immediate nerve fusion (i.e., 24 weeks after the initial nerve transection), greater nerve and muscle electrophysiological functional recovery was observed in the TE-NMI group (Figures 5B, 5C). Also, faster conduction velocities were observed in animals that had undergone TE-NMI prior to non-immediate nerve repair compared to acellular controls. Furthermore, evoked muscle responses were elevated in the TE-NMI group compared to the acellular group. These electrophysiological data indicate that at 1 month after non-immediate nerve repair, the TE-NMI group had greater functional recovery, including nerve conduction and muscle reinnervation, compared to acellular controls.

[0082] To assess regeneration at 4 weeks after repair, cross-sectional nerve morphometric analysis was completed to identify Schwann cells, host axons / fused axons, and myelin (Figure 6A, 6B). There was no difference in the number of host axons distal to the repair site (Figure 6C), but larger host axons were seen in the TE-NMI group (Figure 6D). Larger Schwann cell expression was also seen in the TE-NMI group (Figure 6E). At 1 month after non-immediate nerve fusion, muscle cross sections were stained for acetylcholine receptors (bungarotoxin) to identify neuromuscular junctions (NMJs), and for the presynaptic marker synaptophysin (Figure 6F, 6G). No significant differences in the total number of acetylcholine receptors (AchRs) were seen in the target muscles (Figure 6H), but a higher percentage of mature NMJs co-labeled for AcHR and synaptophysin were observed after TE-NMI implantation (Figure 6I). Furthermore, increased muscle weight was observed in the TE-NMI group compared with controls (data not shown). Collectively, these findings support the histological data and suggest that TE-NMI accelerates ongoing regeneration, maturation, and reinnervation following non-immediate nerve repair.

[0083] In this study, TE-NMI was developed as a novel implantable microtissue featuring a preformed neural network composed of distinct populations of motor and sensory neurons spanning bundled axonal tracts. After transplantation into transected rat nerves, we found that TE-NMI neurons extended numerous axons deep within the host tissue, interacted closely with endogenous Wüngner's zone, and elicited a greater Schwann cell response compared to controls. Furthermore, we show that TE-NMI grafts promote functional recovery after non-immediate nerve repair by preserving a pro-regenerative environment in the distal nerve. Taken together, we report TE-NMI as the first artificial microtissue designed to prevent the deleterious effects of long-term denervation by providing a local source of axons to innervate otherwise denervated muscles.

[0084] Although slow axonal regeneration (1–2 mm / day) is often cited as the primary challenge to successful functional recovery after nerve injury, two more important and often underappreciated factors are (1) the ability of Schwann cells to support injured proximal neurons and promote axonal regrowth, and (2) the receptivity of distal muscles to reinnervation. Prolonged periods of no axonal contact in distal nerves and muscles are common clinical occurrences, often occurring in delayed nerve repair, repair of proximal nerve injuries, and / or repair of long-slit nerve injuries. In these cases, prolonged denervation results in the loss of the regenerative environment and target muscle receptivity necessary for successful regeneration and reinnervation.

[0085] To date, there are no commercially available strategies designed to “babysit” or preserve the regenerative capacity of distal nerves. Innovative surgical techniques have been proposed, such as supercharged end-to-side (SETS) nerve transfer, which reroutes axons to denervated distal nerves, either close to their final target or far to the primary repair. Indeed, SETS may improve functional recovery in difficult nerve repairs by allowing early reinnervation by axons far from the primary repair site (more distal). However, nerve transfer is only indicated in certain scenarios and requires the transection of otherwise healthy nerves, increasing the risk of painful neuroma formation. Therefore, TE-NMI may be more desirable as a more broadly applicable tissue engineering-based approach to “babysitting” that preserves the regenerative capacity of Schwann cells in distal nerves as well as target muscles, without deliberately transecting uninjured nerves.

[0086] TE-NMI is the first preformed microtissue designed to improve functional recovery after nerve repair. These efforts build on previous studies showing that ectopic neurons grafted into distal nerves can preserve the regenerative capacity of Schwann cells and muscles and promote functional recovery after non-immediate nerve repair. Furthermore, previous studies by our group have also shown that tissue engineered nerve grafts (TENGs), which are outgrowth living scaffolds composed of neurons and long axonal tracts, extend neurite processes into otherwise denervated nerves. TENGs simultaneously promote axonal regeneration across challenging defects while preserving regenerative capacity within the distal nerve. Although this dual mechanism remains promising for bridging repair, nerve transection is required for implantation, and distal nerve preservation requires the destruction of otherwise intact distal nerve structures. Therefore, TE-NMI was developed as a next-generation preservation strategy suitable for minimally invasive delivery.

[0087] Nerve fusion has been well described by Bittner et al. as a novel technique to immediately restore axonal membrane continuity and electrical conduction across the junction site after repair. These studies also reported that nerve fusion prevents Wallerian degeneration, minimizes muscle atrophy, and promotes reinnervation, which together result in rapid behavioral recovery. Although the possibility of nerve fusion remains intriguing, it is currently limited to acute nerve injuries where the inevitable Wallerian degeneration causes distal axonal degeneration that precludes fusion. In this study, we present the first example of non-immediate nerve fusion with exogenous TE-NMI axons in an otherwise denervated distal sheath. Successful fusion was achieved by sacrificing the TE-NMI immersed in hypotonic saline, allowing for immediate reconnection between the newly axotomized distal extrinsic axon and the proximal stump. Although immediate electrical conduction was obtained, it is unclear whether the axons remain fused long-term after fusion. Indeed, at 1 month after fusion, the TE-NMI group had higher functional recovery compared to cell-free controls, but a clear decline was observed compared to early recovery. It is possible that the fused axons were broken or removed during the maturation process. Although this study aimed to demonstrate feasibility, future studies should include longer time points to evaluate functional recovery after non-immediate nerve repair. Here, we show that fusion with exogenous TE-NMI axons in otherwise denervated distal stumps allows for greater functional recovery, neural maturation, and muscle reinnervation after non-immediate nerve repair; however, additional studies are needed to elucidate whether these effects are simply due to delayed nerve fusion or delayed nerve repair. Future studies will look at technology transfer with additional efficacy testing using human-compatible cell sources in large animal models.

[0088] Another important concept to address is that this study demonstrates that tissue engineered neural constructs can integrate with denervated muscles. Innervation plays a key role in development and has been shown to be crucial during the biofabrication process of tissue engineered end organs or muscle scaffolds. Furthermore, future studies may include using TE-NMI as an adjunct strategy to enhance tissue biofabrication or for other regeneration strategies that require exogenous axons, such as volumetric muscle loss. Additional optimization may result in even higher functional recovery, for example, supplementing TE-NMI with preformed aligned Schwann cells may enhance motor neuron viability.

[0089] Based on these findings, TE-NMI may be a novel approach for peripheral nerve restoration surgery that allows foreign axons to provide early muscle reinnervation to enhance the chances of successful recovery after non-immediate nerve repair. Furthermore, foreign axons may be spliced ​​with the host nerve, thus enabling non-immediate nerve fusion. Taken together, TE-NMI offers surgeons an opportunity to improve functional recovery and may restore hope to patients with injuries that are currently not suitable for nerve transfer surgery.

[0090] The disclosures of any and all patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A tissue-engineered neuromuscular interface (TE-NMI) fabricated by tissue engineering, comprising: An extracellular matrix core, wherein the extracellular matrix core contains a population of neurons at a first end of the extracellular matrix core, and the population of neurons has axons extending at least partially along the extracellular matrix core, the population of neurons being selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons, the extracellular matrix core and the tissue-engineered neuromuscular interface (TE-NMI) fabricated by tissue engineering.

2. A second population of neurons at a second end of the extracellular matrix core, having axons extending at least partially along the extracellular matrix core, being selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons the second population of neurons and further comprising the TE-NMI according to claim 1.

3. The TE-NMI according to claim 1 or claim 2, having a maximum cross-sectional dimension selected from the group consisting of about 10 μm to about 25 μm, about 25 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 400 μm, about 400 μm to about 500 μm, about 500 μm to about 700 μm, about 700 μm to about 1000 μm, about 1000 μm to about 1500 μm, about 1500 μm to about 2000 μm, about 2000 μm to about 2500 μm, and about 2500 μm to about 3000 μm.

4. The TE-NMI according to claim 3, wherein the extracellular matrix core includes a hydrogel sheath coaxially surrounding the extracellular matrix core, and the hydrogel sheath has a maximum cross-sectional dimension selected from the group consisting of about 20 μm to about 50 μm, about 50 μm to about 100 μm, about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, about 800 μm to about 1200 μm, about 1200 μm to about 1700 μm, about 1700 μm to about 2200 μm, about 2200 μm to about 2700 μm, and about 2700 μm to about 3200 μm.

5. The TE-NMI according to claim 4, wherein the hydrogel sheath has a maximum cross-sectional dimension of about 701 μm and the extracellular matrix core has a maximum cross-sectional dimension of about 300 μm.

6. The TE-NMI according to claim 1 or claim 2, having a length of about 100 μm to about 200 μm, about 200 μm to about 250 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 600 μm, about 600 μm to about 800 μm, about 800 μm to about 1200 μm, about 1200 μm to about 1500 μm, and about 1500 μm to about 2000 μm.

7. one or more non-neuronal cells selected from the group consisting of endothelial cells, muscle cells, myoblasts, astrocytes, olfactory ensheathing cells, oligodendrocytes or Schwann cells The TE-NMI according to claim 1 or 2, further comprising

8. The TE-NMI according to claim 1 or 2, wherein the neurons are derived from stem cells or isolated from dorsal root ganglia.

9. The TE-NMI according to claim 1 or 2, wherein the neurons are heterologous neurons, autologous / patient-specific neurons, homologous neurons, whole dorsal root ganglia or sensory explants.

10. The TE-NMI according to claim 1 or 2, wherein the neurons are heterologous neurons derived from wild-type or transgenic pigs.

11. The TE-NMI according to claim 1 or 2, wherein the extracellular matrix core comprises collagen, gelatin, laminin, fibrin, fibronectin and / or hyaluronic acid.

12. The TE-NMI according to claim 4, wherein the hydrogel sheath comprises agarose, collagen, gelatin, silk, chitosan, fibrin and / or hyaluronic acid.

13. A tissue-engineered neuromuscular interface (TE-NMI) for use in a method of preserving the regenerative capacity of distal nerve segments after peripheral nerve injury in a subject in need thereof, the method comprising transplanting a tissue-engineered neuromuscular interface (TE-NMI) to the distal site of a distal nerve segment, The TE-NMI comprising: an extracellular matrix core, the extracellular matrix core comprising a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons that extend along at least a portion of the extracellular matrix core, The neuronal population is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core comprising A tissue-engineered neuromuscular interface (TE-NMI).

14. The TE-NMI is as follows: A second population of neurons at the second end of the extracellular matrix core, having axons that extend along at least a portion of the extracellular matrix core, selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons The second population of neurons The TE-NMI for use according to claim 13, further comprising.

15. The TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed immediately after the injury.

16. The TE-NMI for use according to claim 15, wherein the injury is caused by surgery.

17. The TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed within less than 24 hours after the injury.

18. The TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed within less than 7 days after the injury.

19. The TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed within less than 2 weeks after the injury.

20. A TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed within less than 1 month after the injury.

21. A TE-NMI for use according to claim 13 or claim 14, wherein the transplantation is performed 1 month or more after the injury.

22. A TE-NMI for use according to claim 13 or claim 14, wherein one or more TE-NMIs are implanted end-to-side into a distal nerve segment, implanted into a nerve bundle, implanted continuously, or implanted into a denervated muscle.

23. A TE-NMI for use according to claim 13 or claim 14, wherein the implantation of one or more TE-NMIs is ultrasound-guided or MRI-guided.

24. A TE-NMI for use according to claim 13 or claim 14, wherein at least two tissue-engineered nerve-muscle interfaces are implanted into a distal nerve segment.

25. A TE-NMI for use according to claim 13 or claim 14, wherein at least five tissue-engineered nerve-muscle interfaces are implanted into a distal nerve segment.

26. A TE-NMI for use according to claim 13 or claim 14, wherein at least ten tissue-engineered nerve-muscle interfaces are implanted into a distal nerve segment.

27. A TE-NMI for use according to claim 13 or claim 14, further comprising performing a primary nerve repair procedure to treat the peripheral nerve injury.

28. A TE-NMI for use according to claim 27, wherein the primary nerve repair procedure includes direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or transplantation of a tissue-engineered nerve.

29. A tissue-engineered neuromuscular interface (TE-NMI) for use in a method of treating peripheral nerve injury in a subject in need thereof, the method comprising the following steps: Transplanting a tissue-engineered neuromuscular interface (TE-NMI) to a distal site of a distal nerve segment, wherein the TE-NMI comprises: An extracellular matrix core, wherein the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, the population of neurons having axons that extend at least in part along the extracellular matrix core, wherein the population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons, the extracellular matrix core comprising, the transplanting step; Monitoring exogenous axonal growth across the entire denervated distal segment as it is, for reinnervation of muscle and / or sensory end organs; Removing one or more tissue-engineered neuromuscular interfaces in the distal nerve segment; and Performing a primary nerve repair procedure, thereby treating the peripheral nerve injury, the performing step comprising, A tissue-engineered neuromuscular interface (TE-NMI).

30. The TE-NMI comprises: A second population of neurons at a second end of the extracellular matrix core, having axons that extend at least in part along the extracellular matrix core, Selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons The second population of neurons A TE-NMI for use according to claim 29, further comprising

31. A TE-NMI for use according to claim 29 or claim 30, wherein the primary nerve treatment comprises transplantation of a nerve graft created by direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or tissue engineering

32. A TE-NMI for use according to claim 29 or 30, wherein the TE-NMI is removed less than 1 week after transplantation

33. A TE-NMI for use according to claim 29 or 30, wherein the TE-NMI is removed less than 1 month after transplantation

34. A TE-NMI for use according to claim 29 or 30, wherein the TE-NMI is removed less than 1 year after transplantation

35. A TE-NMI for use according to claim 29 or 30, wherein the TE-NMI is removed 1 year or more after transplantation

36. A tissue-engineered neuromuscular interface (TE-NMI) for use in a method of treating peripheral nerve injury in a subject in need thereof, the method comprising the following steps: Transplanting a tissue-engineered neuromuscular interface (TE-NMI) to the distal site of a distal nerve segment, wherein the TE-NMI comprises: An extracellular matrix core, wherein the extracellular matrix core comprises a population of neurons at a first end of the extracellular matrix core, and the population of neurons has axons that extend at least a portion along the extracellular matrix core The population of neurons is selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons. The extracellular matrix core The step of implanting, comprising the same; Monitoring exogenous axonal growth throughout the entire denervated distal segment that would otherwise be intact, for innervation of muscle and / or sensory end organs. Removing one or more tissue-engineered neuromuscular interfaces made in the distal nerve segment; and Fusing the TE-NMI axons in the distal nerve segment with at least one proximal axon Comprising A tissue-engineered neuromuscular interface (TE-NMI).

37. The TE-NMI is as follows: A second population of neurons at the second end of the extracellular matrix core, Having axons that extend at least in part along the extracellular matrix core, Selected from the group consisting of one or more motor neurons, one or more motor neurons co-cultured with one or more sensory neurons, and co-aggregates comprising one or more motor neurons and one or more sensory neurons The second population of neurons The TE-NMI for use according to claim 36, further comprising the same.

38. The TE-NMI for use according to claim 36 or claim 37, wherein a reagent is applied prior to removing the TE-NMI to inhibit axonal degeneration.

39. The TE-NMI for use according to claim 38, wherein the reagent comprises hypotonic saline or a calcium chelator.

40. A TE-NMI for use according to claim 39, wherein the reagent is a hypotonic physiological saline solution containing a calcium chelating agent.

41. A TE-NMI for use according to claim 36 or 37, wherein primary nerve repair is performed.

42. A TE-NMI for use according to claim 41, wherein the primary nerve treatment includes direct anastomosis, autograft, allograft, nerve conduit, nerve transfer, or transplantation of a nerve graft produced by tissue engineering.

43. A TE-NMI for use according to claim 41, wherein a free radical scavenger is applied before primary nerve repair.

44. A TE-NMI for use according to claim 43, wherein the free radical scavenger is methylene blue.

45. A TE-NMI for use according to claim 41, wherein a fusion agent is applied during primary nerve repair to promote membrane sealing.

46. A TE-NMI for use according to claim 45, wherein the fusion agent is polyethylene glycol or chitosan.

47. A TE-NMI for use according to claim 45, wherein the application of the fusion agent promotes nerve regeneration and functional recovery.

48. A TE-NMI for use according to claim 13, 14, 29, 30, 36, or 37, further comprising a hydrogel sheath coaxially surrounding the extracellular matrix core.

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